Cultivation and separation of hydrocarbon consuming micro-organisms



United States Patent US. Cl. 195--28 23 Claims ABSTRACT OF THE DISCLOSURE Cultivation of a micro-organism in the presence of a hydrocarbon feedstock consisting wholly or in part of straight chain hydrocarbons, in the presence of an aqueous nutrient medium and in the presence of a gas con taining free oxygen, thereafter maintaining, in a purification stage, said micro-organism with an aqueous nutrient medium and a gas containing free oxygen whereby the hydrocarbon contaminating the micro-organism is reduced in quantity, thereafter subjecting the product containing the micro-organism to a separation stage, thereafter recovering a fraction containing the mirco-organism, thereafter subjecting the fraction containing the microorganism to solvent extraction and thereafter drying the fraction containing the microorganism obtained from the solvent extraction stage.

This invention relates to a process for the production of micro-organisms, for example, yeasts. This invention also relates to a process for the removal of straight chain hydrocarbons, wholly or in part, from mixtures of said hydrocarbons with other hydrocarbons.

In a process in which a micro-organism is cultivated in the presence of a hydrocarbon it has now been found that the micro-organism may be contaminated by sorbed hydrocarbons. Sometimes other materials formed in the fermentation process may be present, thus yeasts may be contaminated by lipids.

In accordance with the present invention there is provided a process which comprises cultivating, in a growth stage, a micro-organism in the presence of a hydrocarbon feedstock consisting wholly or in part of straight chain hydrocarbons, in the presence of an aqueous nutrient medium and in the presence of a gas containing free oxygen, thereafter maintaining, in a purification stage, said microorganism with an aqueous nutrient medium and a gas containing free oxygen whereby the hydrocarbon contaminating the micro-organism is reduced in quantity, thereafter subjecting the product containing the microorganism to a separation stage, thereafter recovering a fraction containing the micro-organism, thereafter subjecting the fraction containing the microorganism to solvent extraction and thereafter drying the fraction containing the micro-organism obtained from the solvent extraction stage.

If desired the product containing the micro-organism obtained by drying may be subjected to further solvent extraction.

The separation stage, referred to hereinbefore, may be a decantation; additionally or alternatively it may be a centrifuging.

Solvents which may be employed in accordance with the invention include ethyl alcohol, iso-propanol, light hydrocarbons, including benzene and light platformate fractions, ethyl ether, acetone, chlorinated solvents and 3,520,777 Patented July 14, 1970 liquefied petroleum gases, such as butane and propane.

Preferably the hydrocarbon feedstock contains hydrocarbon of C or higher. Suitably there may be used a hydrocarbon fraction derived from petroleum.

It is wellknown that certain petroleum fractions, particularly gas oils, contain straight chain hydrocarbons, mainly paraffins which are waxes and which have an adverse effect upon the pour point of the fraction; that is to say, when these hydrocarbons are removed, wholly or in part, the pour point of the fraction is lowered. Usually the wax is removed by precipitation by means of solvents, the wax originally present in the fraction being recovered as such, that is, without conversion to more valuable products.

The petroleum fractions boiling below the gas oils, for example, heavy naphthas and kerosines also contain straight chain hydrocarbons which are potentially valuable for conversion to other products but hitherto, in general, utilisation of these hydrocarbons has been rendered difficult by the necessity of recovering these hydrocarbons from the petroleum fractions, in which they are contained, before they can be converted to other products.

According to a preferred feature of this invention there is provided a process which comprises cultivating a microorganism in the manner as hereinbefore described in the presence of a petroleum fraction consisting in part of straight chain hydrocarbons and having a mean molecular weight corresponding to at least 10 carbon atoms per molecule, and in the presence of an aqueous nutrient medium; and in the presence of a gas containing free oxygen and separating from the mixture, on the one hand, the micro-organism and, on the other hand, a petroleum fraction having a reduced proportion of straight chain hydrocarbons or which is free of said straight chain hydrocarbons.

The process of the invention is of particular value for the treatment of petroleum gas oil fractions which contain straight chain hydrocarbons in the form of waxes, since by the process of the invention, a gas oil of improved pour point is obtained while the waxes are converted to a valuable product.

Usually the straight chain hydrocarbons will be present in the feedstocks according to the invention as paraflins; however, the straight chain hydrocarbons may be present as olefins; also there may be used a mixture containing straight chain parafiins and olefins.

It is an important feature of this invention that when cultivating yeasts in the presence of the feedstocks hereinbefore described under conditions favouring the growth of the yeasts at the expense of the straight chain hydrocarbons, the other hydrocarbons, for example iso-paraffins, naphthenes and aromatics are not metabolised or, at most, the proportion which is metabolised is very small. Furthermore, unlike conventional chemical processes governed by the law of mass action, the rate of removal of straight chain hydrocarbons is not substantially reduced as the proportion of these hydrocarbons in the overall mixture of hydrocarbons decreases (except, of course, in the very final stages of removal). Thus, when desired, the percentage conversion of straight chain hydrocarbons which is achieved can be maintained at a value approaching without necessitating a very disproportionate expenditure of contact time to achieve small improvements. Furthermore, in the continuous process, this high percentage conversion can be achieved without resorting to the use of a long reaction path.

By the application of this process under conditions which limit the metabolism of the straight chain hydrocarbons it is possible to operate with the removal of only a desired proportion of these hydrocarbons.

Suitable feedstocks to the process of the invention inelude kerosine, gas oils and lubricating oils; these feedstocks may be unrefined or may have undergone some refinery treatment, but will usually be required to contain a proportion of straight chain hydrocarbons in order to fulfill the purpose of this invention. Suitably the petroleum fraction will contain 3-45 by weight of straight chain hydrocarbons.

Micro-organisms which are cultivated as herein de scribed may be yeasts, moulds or bacteria.

Preferably when a yeast is employed this is of the family Cryptococcaceae and particularly of the sub-family Cryptococcoideae; however, if desired there may be used, for example, ascosporogeneous yeasts of the sub-family Saccharomycoideae. Preferred genera of the Cryptococcoideae sub-family are Torulopsis (also known as Torula) and Candida. Preferred species of yeast are as follows. In particular it is preferred to use the specific stock of indicated reference number; these reference numbers refer to CBS stock held by the Central Bureau vor Schimmelculture, Baarn, Holland and to INRA stock held by the Institut National de la Recherche Agronomique, Paris, France.

Preferred strain Candida lipolytica.

Candida pulcherrima CBS 610 Candida utilis. Candida utilis, Variati major CBS 841 Candida tropicalis CBS 2317 Torulopsis c lliculosa CBS 133 Hansenula anomala CBS 110 Oidium lactis.

Neurospora sitophila.

Mucoderma cancoillote INRA: STV 11 Of the above Candida lipolytica is particularly preferred.

If desired the microorganism may be a mould. Penicillium expansum is suitable for cultivation in an aqueous nutrient medium containing hydrocarbons.

Penicillium roquefortii, Penicillium nolatum, Aspergillus fussigatus, Aspergillus niger and Asp'ergillus versicOI r may be used for cultivation on a solid agent containing hydrocarbons as feedstock.

Suitably the bacteria are of one of the orders:

Pseudomonadales, Eubacteriales and Actinomycetales.

Preferably the bacteria which are employed are of the family Bacillaceae and Pseudomonadaceae. Preferred species are Bacillus megaterium, Bacillus subtilis and Pseudomonas aeruginosa.

Other species which may be employed include:

Bacillus amylobacter Pseudomonas natriegens Arthrobacter sp. Micrococcus sp. Corynebacterium sp. Pseudm nas syringae Xanthomonas begoniae Flavobacterium devorans Acetobacter sp. Actinomyces sp.

For the growth of the micro-organism it will be necessary to provide, in addition to the feedstock, an aqueous nutrient medium and a supply of oxygen, preferably in the form of air.

A typical nutrient medium for the growth of Nocardia, a genus in the Actinomycetales order, has the following composition:

Grams Ammonium sulphate 1 Magnesium sulphate 0.20 Ferrous sulphate, 7H O 0.005 Manganese sulphate, 1H O 0.002 Monopotassium phosphate 2 Disodium phosphate 3 Grams Calcium chloride '0.1 Sodium carbonate 0.1 Yeast extract 0.008

Distilled water (to make up to 1000 mls.).

For other bacteria a suitable nutrient medium has the the composition:

Monopotassium phosphate7 grams Magnesium sulphate, 7H O0.2 gram Sodium chloride-0.1 gram Ammonium chloride2.5 grams Tap Water (trace elements)-- mls. Yeast extract-0.025 gram Made up to 1000 mls. with distilled water.

A suitable nutrient medium for yeasts (and moulds) has the composition:

Grams Diammonium phosphate 2 Potassium chloride 1.15 Magnesium sulphate, 7H O 0.65 Zinc sulphate 0.17 Manganese sulphate, 1H O 0.045 Ferrous sulphate, 7H O 0.068 Tap water 200 Yeast extract 0.025

Distilled water (to make up to 1000 mls.).

Micro-organisms, and in particular yeasts, when first cultivated with the use of hydrocarbon fractions as feedstock sometimes grow with difiiculty and it is sometimes necessary to use an inoculum of a micro-organism which has previously been adapted for growth on the hydrocarbon fraction which it is intended to use. Furthermore the micro-organism although cultivated in the presence of an aqueous mineral medium containing the appropriate nutrient elements may grow with difficulty, because the hydrocarbon fraction does not contain the growth factors which exist in carbohydrate feedstocks, unless these growth factors are added.

The growth of the micro-organism used is favoured by the addition to the culture medium of a very small proportion of extract of yeast (an industrial product rich in vitamins of group B obtained by the hydrolysis of a yeast) or more generally of vitamins of group B and/or biotin. This quantity is preferably of the order of 25 parts per million with reference to the aqueous fermentation medium. It can be higher or lower according to the conditions chosen for the growth.

The growth of the micro-organism takes place at the expense of the feedstock fraction with the intermediate production of bodies having an acid function, principally fatty acids, in such manner that the pH of the aqueous mineral medium progressively diminishes. If one does not correct it the growth is fairly rapidly arrested and the concentration of the micro-organism in the medium, that is cellular density, no longer increases so that there is reached a so-called stationary phase.

Preferably therefore the aqueous nutrient medium is maintained at a desired pH by the step-wise or continuous addition of an aqueous medium of high pH value. Usually, when using moulds or yeasts, and in particular when using Candida lip lytica, the pH of the nutrient medium will be maintained in the range 3-6 and preferably in the range 4-5. (Bacteria require a higher pH, usually 6.5-8.) Suitable alkaline materials for addition to the growth mixture include sodium hydroxide, potas sium hydroxide, disodium hydrogen phosphate and ammonia, either free or in aqueous solution.

The optimum temperature of the growth mixture will vary according to the type of micro-organism employed and will usually lie in the range 2535 C. When using 2C1n3d2ida lipolytica the preferred temperature range is The take-up of oxygen is essential for the growth of the micro-organism. The oxygen will usually be provided as air. In order to maintain a rapid rate of growth the air, used to provide oxygen, should be present in the form of fine bubbles under the action of stirring. The air may be introduced through a sintererd surface. However there may be used the system of intimate aeration known as vortex aeration.

It has been found that by the use of yeast of the strain Candida lipolytica in a process according to the invention in which aeration is effected by vortex aeration, a high growth rate is achieved whereby the generation time lies in the range 25 hours and the cell concentration is increased by a factor of up to 1000 in two days.

In batch operation, the micro-organism will usually grow initially at a low rate of increase in cellular density. (This period of growth is referred to as the lag phase) Subsequently the rate of growth will increase to a higher rate of growth; the period at the higher rate of growth is referred to as the exponential phase and subsequently again the cellular density will become constant (the stationary phase).

A supply of the micro-organism for starting the next batch will preferably be removed before the termination of the exponential phase.

The growth operation will usually be discontinued before the stationary phase.

At this stage the micro-organism will usually be separated from the bulk of the un-used feedstock fraction.

According to one method of treating the product the major part of the continuous aqueous phase is first separated; preferably this is carried out by centrifuging, or decanting. The separated aqueous phase will usually contain a greater concentration of non-nutritive ions than can be tolerated in the recycle stream and when this is so, only a proportion of the recovered aqueous phase can be recycled. Thus it will usually be possible to separate ca. 96% by wt. of the aqueous phase which is present in the product, of which on the same percentage basis, ca. 20% by wt. will be discarded. The recycle stream is supplied with make-up quantities of the necessary nutrients and is returned to the fermenter; if desired the makeup materials may be fed to the fermenter as a separate stream.

By centrifuging the product from the fermenter (preferably after decanting as described) three fractions are recovered. These are in order of increasing density:

i) an oil phase containing micro-organism cells (ii) an aqueous phase containing traces of oil and microorganism, and

(iii) a micro-organism cream consisting of micro-organism, having a quantity of oil fixed on to the cells, together with aqueous phase.

Fraction (iii) will then be mixed with an aqueous nutrient medium, which may be the same as or different from the aqueous nutrient medium employed in the growth stage, and is maintained in admixture with a gas containing free oxygen whereby the hydrocarbon contaminating the micro-organism is reduced in quantity or is eliminated.

Again, preferably, the major part of the continuous aqueous phase is separated with recycle of a proportion of the recovered aqueous phase, as hereinbefore described; recycle may be to either the growth stage or purification stage as desired.

By centrifuging the product from the fermenter, preferably after separating the major part of the aqueous phase by decanting, there are obtained:

(i) an aqueous phase containing traces of micro-organism (ii) a micro-organism cream.

This micro-organism cream which contains aqueous material may be treated by continuous solvent extraction or by successive washings with solvent followed by phase separation. Preferably the cream contains 1:1 to 3:1 by weight of water to micro-organism (dry weight). If desired a paste may be recovered by centrifuging and diluted with water to form the cream.

Suitably the extraction is carried out in a stationary vessel equipped with paddle stirrers, preferably rotating at less than 10 revs. per minute or in a vessel which rotates on a horizontal axis. When operating a continuous solvent extraction, the extract is withdrawn continuously and distilled, continuously or batchwise, at atmospheric or reduced pressure, and solvent continuously fed back to the extractor. Under these conditions the yeast may be introduced and withdrawn continuously or batchwise.

Preferably the solvent extraction is effected while feeding solvent to the extractor at a periodically varying rate to create pulsations in the flow of said liquid stream.

The pulses of the liquid passing through the solid material bring about oscillations and limited displacements of each grain of solid material in relation to its neighbours, and this is equivalent to a mechanical agitation of the whole. For this reason the whole of the products to be extracted is much more rapid and complete.

Suitably there is arranged in the feed of the liquid stream a device which imparts to it pulses whose amplitude and frequency are regulated experimentally at the most favourable value for each particular case. These pulses are produced by any suitable processes already known, and preferably an alternating pump is used whose valves have been removed.

Preferably the number of pulses lies between 1 and 60 per minute. The operation of the process under the action of pulses is further described in British patent application 2,234/63 (SFP 1404).

Suitable solvents for use in the process have been described hereinbefore. If desired a first extraction stage can be operated using a polar solvent, for example an alcoholic solvent, for example ethanol or isopropanol and then the partially purified microorganism can be further treated in a second extraction stage using a hydrocarbon solvent, for example normal hexane or a light platformate fraction or benzene.

Preferably in the second stage there is used as solvent a mixture of hydrocarbon in major amount with a polar solvent in minor amount. Preferably there is used the azeotropic mixture of hexane with isopropanol or ethanol. If desired both extraction stages can be operated in continuous manner.

When using a solvent consisting of a mixture of hydrocanbon and a polar solvent, it is believed that the function or one function of the polar solvent is to weaken the bonding of the material to be extracted (even the bonding or hydrocarbons which are not themselves soluble in the polar solvent).

If a single washing is employed in the first stage the amount of ethanol or isopropanol which is used should be 1.5-3 times the volume of water which is present in the cream or paste of the micro-organism. However if desired two washings with ethanol or isopropanol may be employed using in the first washing a volume of solvent equal to the volume of water in the cream or paste and in the second washing a smaller amount of ethanol or isopropanol for example, one half of the amount used in the first washing.

Between washings of each stage or sub-stage the cream or paste is allowed to drain, for example by filtering and part of the residual solvent is then preferably removed by vacuum filtration.

In the second stage the amount of solvent used in the (or each) washing will usually be 2-20 times the volume of the resulting dry micro-organism.

By the use in the second extraction stage of a solvent which is a mixture of hydrocarbon and a polar solvent the composition of the second stage solvent, which will in any case acquire polar solvent from the first stage extraction, can be stabilised. Build up of polar solvent can be avoided in the course of a distillation stage, in which the second stage solvent is recovered by the removal of separate streams consisting of (a) polar solvent for recycle to the first extraction stage and (b) a mixture of hydrocarbon and polar solvent for recycle to the second stage. Suitably in a distillation stage the extract obtained by the second extraction stage is distilled to recover (a) overhead a mixture of hydrocarbon, polar solvent and water for recycle to the second extraction stage and (b) a bottoms fraction containing polar solvent, water and the extracted materials; this fraction is preferably blended with the extract obtained in the first extraction stage before this is fed to distillation whereby all contaminants recovered by solvent extraction are removed as a bottoms fraction in this distillation stage. Suitably the polar solvent is ethanol or isopropanol. Suitably the second stage solvent is an azeotropic mixture.

The micro-organism fraction, from which as much solvent as possible has been removed by decantation and/ or centrifuging and/or filtration, is freed from the remaining traces of solvent and/or water by evaporation, preferably under reduced pressure.

If desired the dry micro-organism fraction may be further extracted with a hydrocarbon containing solvent.

A yeast which has been freed from the whole or part of its lipids and the contaminating hydrocarbons by one of the methods described hereinbefore and whose taste has been improved is a new industrial product of value for human nutrition.

The lipid extract which has been recovered by the evaporation of the solvent is also a new industrial product which can be used either as such or as a raw material for the separation of its sterols, fatty acids (either before saponification or after) or of its other constituents.

Any stages of the process may be operated continuously or batch-wise.

When operating continuously it will usually be necessary to provide separate fermenters or separate zones in the same fermenter for the operation of the growth and purification stages.

Other steps which may be taken to obtain a purified micro-organism or a product derived therefrom or to improve the process in respect of the production of the unmetabolised hydrocarbon fraction are described in the following applications; the use of any process herein described lies within the scope of the present invention.

Preferred methods for use in the cultivation of the micro-organism and for the recovery of the product are described in British patent specification Nos. 914,567 and 915,568.

EXAMPLE 1 40 litres of an aqueous mineral nutrient medium having the composition given below were introduced in a (first stage) stainless steel fermenter having an effective capacity of 60 litres.

In order to keep the temperature in the fermenter constant at 30 C., water was circulated in an annulus constituted by the space between two concentrical cylinders, the smaller one being the fermenter itself.

The aqueous nutrient medium had the composition:

Grams Diammonium hydrogen phosphate 2 Potassium chloride 1.15 Magnesium sulphate 7H O 0.65 Zinc sulphate 0.17 Manganese sulphate IH O 0.045 Ferrous sulphate 7H O 0.068 Yeast extract 0.025

Tap water 200 Distilled water add. 1000 ml.

20 litres of a 24 hours inoculum of Candida tropicalis on mixed C -C normal hydrocarbons were then added, such that the cellular density Was about l gram of dry matter per litre.

1.030 litres of heavy gas-oil, that is grams/litre were then added this quantity being sufficient to take the cellular density to 2 grams/litre.

The temperature of the culture was kept at 30:1 C. pH at 4 and aeration and agitation giving 3 millimoles of 0 per litre of medium per minute. Ammonia solution was admitted by an automatic pH controller.

When the flow of ammonia reached ml. the addition of gas-oil was begun assuming a yield on gas-oil of (dry yeast produced) gas-oil feed of 10% by weight and a cell division time of 3 hours. This addition was carried out every hour until 200 grams/litre, i.e. 13.8 litres had been added.

Starting with a cellular density of 2 grams/litre at hours (at the end of the exponential growth phase) the cellular density was 15 grams/litre. The fermenter was then run continuously at a dilution rate of 0.2 vol./vol./ hour, cellular density remaining constant at 15 grams/ litre throughout the run. The broth collected at the outlet of the fermenter was subjected to decanting, an aqueous phase consisting of 65% by weight of the broth being withdrawn as spent medium and the remainder of the broth, in the form now of a yeast cream recovered. This yeast cream had the composition (percent by weight):

Percent Yeast (dry weight) 4.5 Residual gas-oil 31.5 Aqueous medium 64.0

Residual gas-oil, apart from that absorbed on yeast, was removed by means of a Sharples supercentrifuge.

The yeast after centrifuging had the following composition:

Percent by wt.

Moisture 68 Lipids (on dry basis) l8 Unsaponifiables (on dry basis) (containing residual adsorbed oil) This recovered yeast was mixed with nutrient medium to achieve a cream of which cellular density was 15 grams/litre.

This yeast cream was continuously fed to a second fermenter identical to the first stage fermenter and operated under conditions identical to the first stage conditrons except that no additional substrate or aqueous medium was supplied. Aeration was maintained at 3 millimoles of 0 per litre of medium per minute.

Broth was continuously withdrawn and passed to a Sharples supercentrifuge and a yeast paste was recovered.

The yeast had, at this stage, the composition:

Moisture-% by wt. (on dry basis) Lipids-3 by wt. Unsaponifiables10% by wt. Nitrogen-10% by wt.

The water content of the yeast paste was adjusted at a level of 1.5 parts by weight of water per part of dry yeast, yielding a yeast cream. This cream was pumped into an extractor and to it was added an azeotropic mixture, of lsopropanol and normal hexane at a rate of 8 parts of solvent per part of yeast (dry weight). This extraction step was repeated and these two extraction steps were followed by a second extraction stage consisting of one isopropanol wash at a rate of 8 parts of solvent per part of yeast (dry weight).

Solvents were then drawn oil, finally under vacuum. Remaining traces of solvent were stripped, and the yeast dried in superheated steam.

The final composition of yeast product was as follows (on dry basis):

Percent by wt. Moisture Lipids 0.2 Unsaponifiables 0.1 Nitrogen a; 11

After these various extraction stages, solvents were recovered, separately or in mixture, and distilled to give:

(a) an azeotropic mixture of isopropanol and normal hexane (b) an azeotropic mixture of isopropanol and water (c) a residue fraction.

The recovered solvents were recycled to the extraction stages.

The second stage fermenter is seen to constitute a purification stage. In this case no detergent washing was employed to remove absorbed oil.

EXAMPLE 2 The process described in Example 1 was repeated except that the solvent extraction of the yeast cream was carried out as follows:

The yeast cream was pumped into an extractor rotating at revs. per minute on a horizontal axis. To this extractor was added an azeotropic mixture of equal parts by volume of isopropanol and normal hexane at a rate of 8 part of mixture per part by weight of dry yeast, at 60 C. for 30 minutes. The solid material from this first extraction stage was extracted with an azeotropic mixture of IPA-n hexane and this step was repeated 6 times. Then solvent was drawn off, finally under vacuum.

The trace amount of residual solvent was removed by final stripping and the yeast product was dried in super heated steam.

The final composition of yeast product was as follows:

Percent by wt.

Water 5 Lipids 0.2 Unsaponifiables 0.1 Nitrogen 11.5

What we claim is:

1. A process which comprises cultivating, in a growth stage, a micro-organism in the presence of a hydrocarbon feedstock comprising straight chain hydrocarbons, an aqueous nutrient medium and a gas containing free oxygen, thereafter in a purification stage, maintaining said micro-organism with an aqueous nutrient medium and a gas containing free oxygen whereby the hydrocarbon contaminating the micro-organism is reduced in quantity, thereafter subjecting to a separation stage, a product of said purification stage, said product containing the micro-organism, thereafter recovering a fraction containing the micro-organism and a reduced proportion of aqueous phase, thereafter subjecting the fraction to solvent extraction and thereafter drying the fraction containing the micro-organism obtained from the solvent extraction stage.

2. A process according to claim 1 in which the microorganism is a paraffinic hydrocarbon consuming microorganism.

3. A process according to claim 1 in which the microorganism is a yeast.

4. A process according to claim 3 in which the yeast is of the family Cryptococcaceae.

5. A process according to claim 4 in which the yeast is of the subfamily Cryptococcoideae.

6. A process according to claim 5 in which the yeast is of the genus Torulopsis.

7. A process according to claim 5 in which the yeast is of the genus Candida.

8. A process according to claim 7 in which the yeast is Candida lipolylica.

9. A process according to claim 7 in which the yeast is Candida tropicalis.

10. A process according to claim 1 in which the microorganism is a bacterium.

11. A process according to claim 1 in which the separation stage consists at least in part of decantation.

12. A process according to claim 1 in which the separation stage consists at least in part of centrifuging.

13. A process according to claim 12 in which a microorganism paste obtained by centrifuging is mixed with Water to form a cream, said cream being thereafter subjected to solvent extraction.

14. A process according to claim 13 in which water is mixed with the paste to give a ratio of water to dry rnicroorganism in the cream in the range 1:1 to 3:1 by weight.

15. A process according to claim 1 in which the solvent extraction is effected by means of a solvent comprising a hydrocarbon.

16. A process according to claim 15 in which the solvent comprising a hydrocarbon is a mixture of a hydrocarbon and a polar solvent.

17. A process according to claim 16 in which the polar solvent is an alcohol.

18. A process according to claim polar solvent is ethanol.

19. A process according to claim polar solvent is isopropanol.

20. A process according to claim hydrocarbon is normal hexane.

21. A process according to claim 15 in which the hydrocarbon is benzene.

22. A process according to claim 15 in which the hydrocarbon is a light platformate fraction.

23. A process according to claim 16 in which the mixed hydrocarbon and polar solvent is an azeotropic mixture.

17 in which the 17 in which the 15 in which the References Cited UNITED STATES PATENTS 8/1966 Filosa 195-82 8/1966 Champagnat et al 195-82 US. Cl. X.R. l3, 82, 96

UNITED STATES PATENT OFFICE Patent No. 3,52 ,777 Dated y 14, 1970 t Jean Antoine Filosa It is certified that error appears in the above-1dentified patent and that said Letters Patent are hereby corrected as shown below:

Col. 4, line 6, delete "the"; and

Col. 7, line 49, "915,568" should read 914,568

buimlx) {ll-ED SEALED 3:515 1971 A mm 1: mm an Edwar Flmha' 1" Gal-1:51am of Patents Meeting 0mm 

